NHE-RF is a new discovered PDZ motif protein that is a necessary co- factor in cAMP-dependent protein kinase (PKA) inhibition of the renal Na/H exchanger, NHE3. NHE-RF binds to NHERF, to NHE3, and to ezrin suggesting that NHE-RF interacts uniquely with its cellular targets forming signaling-complexes. The present grant focuses on the biochemical requirements for and physiologic significance of the physical interaction between proteins moieties involved in PKA mediated inhibition of NHE3. Specific Aim 1: To determine the functional role of NHE-RF dimerization. NHE-RF binds to NHE-RF forming head-to-head dimers but the mechanism of and the physiologic effect of dimerization are unexplored. Biochemical (Far Westerns) and biophysical (biosensor) assays will be used to define the kinetics of NHE-RF/NHE-RF binding. Yeast two-hybrid screening of mutant mini-libraries will be used to determine the binding amino acid sequences and to select mutants that fail to dimerize. The physiologic effect of dimerization on the effect of cAMP on Na/H exchange will be studied using fluorescence techniques in PS120 cells expressing NHE3 and NHE-RF mutants that do not dimerize. Specific Aim 2: To delineate the molecular basis for NHE3 regulation by NHE-RF. NHE-RF binds to NHE3 and the two proteins co-immunoprecipitate. Biochemical and biophysical assays will be used to define the kinetics of NHE-RF/NHE3 binding, and yeast two-hybrid screening of mutant mini- libraries to determine the binding amino acids sequences and to select loss-of-function mutants. The effect of cAMP on Na/H transport, the phosphorylation state of NHE3, and the co-immunoprecipitation of NHE-RF and NHE3 will be examined in PS120 cells expressing mutant NHE-RF and/or NHE3 which do not bind. Specific Aim 3. To define the physiologic importance of NHE-RF in the PKA regulation of NHE3. New data suggests NHE-RF binds to the PKA anchor protein, ezrin. Biochemical and biophysical assays will be used to study the kinetics of NHE-RF/ezrin binding and yeast two-hybrid studies to define the binding sequences. The physiologic effect of NHE- RF/ezrin binding will be studied in PS120 cells expressing loss-of- function NHE-RF mutants or polypeptides representing the ERM region of ezrin.